Identification and characterization of a novel integrin-linked kinase inhibitor

Article abstract of DOI:10.1021/jm2007744

Integrin-linked kinase (ILK) represents a relevant target for cancer therapy in light of its role in promoting oncogenesis and tumor progression. Through the screening of an in-house focused compound library, we identified N-methyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(4′-(trifluoromethyl)-[1, 1′-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide (22) as a novel ILK inhibitor (IC₅₀, 0.6 μM), which exhibited high in vitro potency against a panel of prostate and breast cancer cell lines (IC₅₀, 1-2.5 μM), while normal epithelial cells were unaffected. Compound 22 facilitated the dephosphorylation of Akt at Ser-473 and other ILK targets, including glycogen synthase kinase-3β and myosin light chain. Moreover, 22 suppressed the expression of the transcription/translation factor YB-1 and its targets HER2 and EGFR in PC-3 cells, which could be rescued by the stable expression of constitutively active ILK. Evidence indicates that 22 induced autophagy and apoptosis, both of which were integral to its antiproliferative activity. Together, this broad spectrum of mechanisms underlies the therapeutic potential of 22 in cancer treatment, which is manifested by its in vivo efficacy as a single oral agent in suppressing PC-3 xenograft tumor growth.

Full text of DOI:10.1021/jm2007744

ARTICLE
pubs.acs.org/jmc
Identification and Characterization of a Novel Integrin-Linked
Kinase Inhibitor
Su-Lin Lee,† En-Chi Hsu,† Chih-Chien Chou,† Hsiao-Ching Chuang,† Li-Yuan Bai,‡ Samuel K. Kulp,†
and Ching-Shih Chen†,*
†Division of Medicinal Chemistry, College of Pharmacy and Comprehensive Cancer Center, The Ohio State University, 336 Parks Hall,
500 West 12th Avenue, Columbus, OH 43210, United States
‡Division of Hematology and Oncology, Department of Internal Medicine, China Medical University, Taichung, Taiwan
ABSTRACT: Integrin-linked kinase (ILK) represents a relevant target for cancer therapy in light
of its role in promoting oncogenesis and tumor progression. Through the screening of an in-
house focused compound library, we identified N-methyl-3-(1-(4-(piperazin-1-yl)phenyl)-
5-(40-(trifluoromethyl)-[1,10-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide (22) as a novel
ILK inhibitor (IC50, 0.6 μM), which exhibited high in vitro potency against a panel of prostate
and breast cancer cell lines (IC50, 1ꢀ2.5 μM), while normal epithelial cells were unaffected.
Compound 22 facilitated the dephosphorylation of Akt at Ser-473 and other ILK targets,
including glycogen synthase kinase-3β and myosin light chain. Moreover, 22 suppressed the
expression of the transcription/translation factor YB-1 and its targets HER2 and EGFR in PC-3
cells, which could be rescued by the stable expression of constitutively active ILK. Evidence indicates that 22 induced autophagy and
apoptosis, both of which were integral to its antiproliferative activity. Together, this broad spectrum of mechanisms underlies the
therapeutic potential of 22 in cancer treatment, which is manifested by its in vivo efficacy as a single oral agent in suppressing PC-3
xenograft tumor growth.
’ INTRODUCTION
mediate the phosphorylation of a variety of signaling proteins,
including Akt at Ser-473, glycogen synthase kinase (GSK)3β,5ꢀ7,13
and myosin light chain (MLC).14,15 Moreover, ILK overexpression
has been associated with the oncogenesis and tumor progression of
many types of malignancies, including those of prostate,16 ovary,17
breast,18 colon,19 pancreas,20 stomach,21 and liver.22 In light of its
role in regulating diverse cellular events, including cell proliferation,
survival, angiogenesis, motility, and epithelialꢀmesenchymal transi-
tion, ILK represents a relevant target for cancer therapy.9 In this study,
we report the identification and validation of a novel ILK inhibitor 22,
which exhibits high potency in suppressing the viability of a panel of
prostate and breast cancer cells (IC50, 1ꢀ2.5 μM) via autophagy and
apoptosis. Our data indicate that this antiproliferative effect was, at
least in part, attributable to the inactivation of Akt signaling and the
transcriptional repression of the transcription factor Y-box binding
protein (YB)-1 and its targets, including HER2 and EGFR. Equally
important, daily oral 22 at 25 and 50 mg/kg was effective in
suppressing PC-3 xenograft tumor growth in nude mice.
The phosphatidylinositol-3-kinase (PI3K)/Akt signaling axis
plays a pivotal role in regulating multiple cellular events includ-
ing cell growth, survival, metabolism, and motility through the
modulation of a plethora of downstream effectors. In response to
growth factor or cytokine stimulation, activated PI3K facilita-
tes the production of phosphatidylinositol 3,4,5-trisphosphate
(PIP3), leading to the membrane recruitment and subsequent
activating phosphorylation of Akt at Thr-308 and Ser-473 by
phosphoinositide-dependent kinase (PDK)1 and PDK2, respec-
tively. In contrast to the well-characterized PDK1,1 the molecular
identity of PDK2 remains elusive.2 Although recent evidence has
suggested that the rictorꢀmTOR complex (mTORC2) acts as a
PDK2,3,4 a number of other kinases have also been implicated in
mediating Akt-Ser-473 phosphorylation,2 one of which is integ-
rin-linked kinase (ILK).5ꢀ7
ILK was originally identified as a binding partner of the cytoplas-
mic tail of integrin β1 through a yeast two-hybrid screening.8 The ILK
protein comprises three major domains, namely the N-terminal
ankyrin repeats, a central pleckstrin homology domain, and C-term-
inal kinase domain.9 Each of these domains plays a role in mediating
ILK’s biological functions through interaction with intracellular
signaling proteins or PIP3. For example, ILK physically interacts with
the cytoplasmic proteins PINCH and parvin to form the ternary
ILKꢀPINCHꢀparvin complex, which serves as a molecular scaf-
fold linking integrins with actin cytoskeleton10 and the microtubule
network11 to modulate actin polymerization and mitotic spindle
orientation, respectively. Although whether ILK contains a functional
kinase domain remains a contentious issue,12 ILK has been shown to
’ CHEMISTRY
In the course of developing different kinase inhibitors, the
authors’ laboratory had used the scaffold of 4-(5-aryl-3-(trifluo-
romethyl)-1H-pyrazol-1-yl)aniline to generate an array of deri-
vatives,23,24 designated as series AꢀC (Figure 1). After preli-
minary modeling analysis by docking these compounds into the
ATP binding pocket of ILK to assess binding affinities, additional
Received: June 14, 2011
Published: August 08, 2011
r
2011 American Chemical Society
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Figure 1. Chemical structures of 1ꢀ53 in the focused compound library used for the biomolecular screening of ILK inhibitors.
derivatives were synthesized by replacing the CF3ꢀ side chain
with a ꢀCONHCH3 (series D and N-methyl-1-(4-(piperazin-1-
yl)phenyl)-5-(40-(trifluoromethyl)-[1,10-biphenyl]-4-yl)-1H-pyr-
azole-3-carboxamide (21)) or ꢀCH2CH2CONHCH3 moiety
(series E and F, and N-methyl-3-(1-(4-morpholinophenyl)-
5-(40-(trifluoromethyl)-[1,10-biphenyl]-4-yl)-1H-pyrazol-3-yl)pro-
panamide (53)) to enhance hydrophilic interactions/hydrogen
bonding with the peptide backbone of the binding site. General
procedures for the syntheses of these new derivatives are depicted
in Scheme 1. Together, these derivatives 1ꢀ53 were used for
the biomolecular screening for ILK inhibitors by Western blot
analysis of the effects of individual agents at 2.5 μM on the
phosphorylation level of Akt at Ser-473 versus Thr-308 in PC-
3 cells.
’ RESULTS
Identification of Putative PDK2 Inhibitors by Screening a
Focused Compound Library. Of the 53 compounds examined,
22 and N-ethyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(40-(trifluoro-
methyl)-[1,10-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide
(23) exhibited strong PDK2 inhibitory activity, i.e. selectivity in
facilitating Akt Ser-473 dephosphorylation without affecting that
of Thr-308, while other agents showed low or no appreciable
activities in mediating Akt dephosphorylation at either residue
(Figure 2A). As these two structurally related compounds differ
only in the N-alkyl moiety, i.e., methyl versus ethyl, 22 was used
as the lead agent for mechanistic validation.
Structureꢀactivity relationship analysis revealed a stringent
structural requirement for the Akt Ser-473 dephosphorylating
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Scheme 1. General Synthetic Procedures for 5, 15ꢀ24, 27ꢀ30, and 53a
a Reaction conditions: (a) 1-(4-bromophenyl)ethanone, Bu4NBr, K2CO3, Pd(OAc)2 (cat.), H2O, 60 °C; (b) diethyl oxalate, NaH, THF; (c) ethyl
4-(1H-benzo[d][1,2,3]triazol-1-yl)-4-oxobutanoate, MgBr2 O(C2H5), CH2Cl2, DIPEA; (d) (4-nitrophenyl)hydrazine hydrochloride, TsOH, EtOH,
3
microwave 130 °C, 10 min; (e) NH2R (R = CH3 or C2H5) in EtOH, 120 °C; (f) H2(g) 70 psi, 10% Pd/C, MeOH/EtOAc; (g) (i) Boc-β-alanine, EDC,
THF, rt; (ii) 3N HCl in MeOH, rt; (h) bis(2-chloroethyl)amine hydrochloride, xylene, 170 °C; (i) 1-iodo-2-(2-iodoethoxy)ethane, xylene, 170 °C.
Figure 2. (A) Identification of putative PDK2 inhibitors by screening of an in-house focused compound library. Western blot analysis of the effects of
1ꢀ53 versus DMSO control on the phosphorylation of Akt on Ser-473 versus Thr-308 in PC-3 cells. Cells were exposed to individual test agents at
2.5 μM or DMSO vehicle in 5% FBS-supplemented RPMI 1640 medium for 24 h. (B) SAR analysis of 22/23 versus the structurally related but PDK2-
inactive derivatives, 5, 14, 6, 21, 18, and 53. As shown, modifications in any of the three peripheral structural motifs, designated as aꢀc, of 22 led to loss of
the Akt Ser-473 dephosphorylating activity, indicating a stringent structural requirement.
activity of 22, for which the integrity of the three peripheral
structural motifs, i.e., 4-trifluoromethyl-biphenyl moiety (motif a),
N-methylpropanamide side chain (motif b), and piperazine ring
(motif c), was critical (Figure 2B). As such, minor modifications
of any of these three motifs resulted in loss of the PDK2
inhibitory activity at 2.5 μM: motif a, substituting the CF3ꢀ
with a ꢀCN (3-(5-(40-cyano-[1,10-biphenyl]-4-yl)-1-(4-(piperazin-
1-yl)phenyl)-1H-pyrazol-3-yl)-N-methylpropanamide (5)) or repla-
cing it with a phenanthrene ring (N-methyl-3-(5-(phenanthren-2-
yl)-1-(4-(piperazin-1-yl)phenyl)-1H-pyrazol-3-yl)propanamide
(14)); motif b, replacing it with a CF3ꢀ moiety (1-(4-
(3-(trifluoromethyl)-5-(40-(trifluoromethyl)-[1,10-biphenyl]-4-
yl)-1H-pyrazol-1-yl)phenyl)piperazine (6)) or shortening the
side chain length by two carbon units (21); motif c, replacing it with
a β-alanine (3-amino-N-(4-(3-(3-(methylamino)-3-oxopropyl)-
5-(40-(trifluoromethyl)-[1,10-biphenyl]-4-yl)-1H-pyrazol-1-yl)-
phenyl)propanamide (18)) or substituting the nitrogen atom
with an oxygen (53).
Compound 22 Exhibits High Antiproliferative Potency.
The antitumor effects of 22 were examined in a panel of prostate
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Figure 3. Effects of 22 on cell viability and ILK signaling. (A) Dose-dependent suppressive effects of 22 on the viability of PC-3 and LNCaP prostate
cancer cells versus PrECs (left panel), and of MDA-MB-231, MDA-MB-468, SKBR3, and MCF-7 breast cancer cells versus MECs (right panel) in 5%
FBS-supplemented medium after 24 h of treatment. Cell viability was determined by MTT assays. Points, means; bars, SD (n = 6). (B) Western blot
analysis of the suppressive effects of shRNA-mediated knockdown of ILK and 22 on the phosphorylation of Akt at Ser-473 and Thr-308 and the
downstream targets of ILK (GSK3β and MLC) versus those of mTORC2 (SGK and PKCR) in PC-3 and/or MDA-MB-231 cells. Cells were exposed to
22 at the indicated concentrations for 24 h in 5% FBS-supplemented medium. The immunoblots shown are representative of three independent
experiments.
and breast cancer cell lines with varying genetic defects and
compared to the effect in normal prostate epithelial cells (PrECs)
and mammary epithelial cells (MEC) by MTT assays (Figure 3A).
Compound 22 differentially suppressed the viability of these
cancer cells with the following IC50 values: prostate cancer cells,
LNCaP, 1.6 μM; PC-3, 2 μM; breast cancer cells: MDA-MB-231,
1 μM; MDA-MB-468, 1.5 μM; SKBR3, 1.8 μM; MCF-7, 2.5 μM.
In contrast, PrECs and MECs were resistant to the antiprolifera-
tive effect of 22 within the dose range of 1ꢀ5 μM.
ILK in an in vitro radiometric kinase assay. Representative auto-
radiographic data from one of several experiments are shown in
Figure 4A, of which the densitometric analysis indicates an IC50
of 0.6 μM. Moreover, the stable expression of GFP-tagged
constitutively active (CA)-ILK in PC-3 cells increased phosphor-
ylation of Ser-473-Akt and GSK3β, while the levels of p-Thr-308-
Akt, p-PKCR, and p-SGK1 remained unaltered (Figure 4B, left
panel). In addition, this overexpression of CA-ILK protected
PC-3 cells from 22-mediated inhibition of cell viability as indi-
cated by MTT assays showing a shift in the doseꢀresponse curve
for CA-ILK-overexpressing PC-3 cells to the right (Figure 4B,
right panel).
Compound 22 Suppressed the Expression of YB-1 and Its
Targets HER2 and EGFR via an ILK-Dependent Mechanism.
Suppression of ILK by either siRNA-mediated knockdown or
pharmacological inhibition has been shown to reduce the expres-
sion of several growth factor receptors, including HER2 and
EGFR,27,28 in breast cancer cells by down-regulating the expres-
sion of the shared transcriptional/translational regulator YB-1.
Pursuant to these findings, we examined the ability of 22 to
modulate the expression of these important signaling effectors in
PC-3 and SKBR3 cells. Western blot and RT-PCR analyses
indicate that 22 reduced the expression of YB-1, HER2, and
EGFR, at both protein and transcript levels, in a dose-dependent
manner in both cell lines (Figure 5A, PC-3; Figure 5B, SKBR3).
Equally important, overexpression of CA-ILK, through stable
and transient transfection in PC-3 and SKBR3 cells, respectively,
Evidence that 22 is an ILK Inhibitor. The PDK2 inhibitory
activity of 22 was confirmed by its dose-dependent suppressive
effect on Akt phosphorylation at Ser-473 without disturbing that
of Thr-308 in PC-3 and MDA-MB-231 cells (Figure 3B). It is
noteworthy that this drug-induced Ser-473-Akt dephosphoryla-
tion was accompanied by parallel decreases in the phosphoryla-
tion levels of GSK3β and MLC, two downstream targets of
ILK, while those of the mTORC2 substrates serum- and gluco-
corticoid-induced protein kinase (SGK)-125 and protein kinase
C (PKC)R26 were unaffected (Figure 3B). As a positive control,
shRNA-mediated knockdown of ILK in PC-3 cells modulated
the phosphorylation of these signaling proteins in a manner
similar to that of 22. Together, these findings suggest that 22
might mediate Ser-473-Akt dephosphorylation through the in-
hibition of ILK.
This premise was corroborated by the dose-dependent in-
hibitory effect of 22 on the phosphorylation of myelin basic
protein (MBP), a known ILK substrate,5 by immunoprecipitated
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diminished the suppressive effect of 22 on these signaling
effectors.
remaining activities of the individual kinases in the profile after
exposure to 5 μM 22 were high (g65%): Abl, 73%; CDK1/
cyclin B, 73%; CDK5/p25, 98%; cKit, 100%; cSRC, 91%; EGFR,
103%; Flt3, 66%; GSK3β, 142%; IKKβ, 102%; Jak2, 114%; Jak3,
128%; Met, 110%; mTOR, 122%; PDK1, 94%; Akt, 88%; PKCR,
97%; Ros, 103%; Rsk1, 65%; ZAP70, 104%. Among the 19
recombinant kinases examined, the only exception was p70S6K,
which exhibited greater than 50% inhibition by 22 (44% residual
activity after treatment). This finding was confirmed by Western
blot analysis of the dose-dependent effects of 22 on the phosphor-
ylation of p70S6K versus its target S6 in PC-3 cells (Figure 6A). As
shown, 22 exhibited a modest suppressive effect on phosphory-
lated S6 levels, without affecting the phosphorylation status of
p70S6K, an mTOR substrate. Moreover, in contrast to the
reported effects of the known ILK inhibitor (E)-4-(substituted-
phenyl)diazenyl-1,3,5-substituted-1H-pyrazole 54 (QLT0267),29
22 did not affect the autophosphorylation of focal adhesion kinase
Specificity in Kinase Inhibition. To assess the specificity of
22’s kinaseinhibitoryactivity, the compound was evaluated against
a panel of 20 recombinant kinases by kinase-profiling assays per-
formed by a commercial vendor (Millipore, Billerica, MA). The
results support a high degree of specificity of 22 for ILK as the
Figure 4. Evidence that 22 is an ILK inhibitor. (A) Dose-dependent
suppressive effect of 22 on the kinase activity of immunoprecipitated
ILK. Kinase activity was determined in the presence of 22 at the
indicated concentrations by measuring 32P-phosphorylation of the
ILK substrate MBP as described in the Experimental Section. Data are
presented as means ( SD (n = 3). (B) Effects of ectopic expression of
GFP-tagged CA-ILK versus GFP alone on the phosphorylation of Akt,
GSK3β, PKCR, and SGK in stable PC-3 transfectants (left panel) and on
the viability of PC-3 cells after 24 h treatment with 22 at different
concentrations in 5% FBS-supplemented medium (right panel). Blot on
the left confirms the presence of the CA-ILK protein (*) in transfected
PC-3 cells. The immunoblots shown are representative of three in-
dependent experiments. Points, means; bars, SD (n = 6).
Figure 6. Specificity of 22 in kinase inhibition. (A) Western blot
analysis of the dose-dependent effect of 22 on the phosphorylation of
p70S6K, S6, Tyr-397-FAK, ERKs, p38, and JNK in PC-3 cells. Cells were
exposed to the indicated concentrations of 22 in 5% FBS-supplemented
medium for 24 h. (B) Effects of the stable expression of CA-ILK, as
depicted in Figure 4B, on 22-mediated dephosphorylation of S6, ERKs,
and p38 in PC-3 cells. The immunoblots shown are representative of
three independent experiments.
Figure 5. Dose-dependent effects of 22 on the protein (left panel) and mRNA (right panel) expression of YB-1 and its targets HER2 and EGFR in (A)
PC-3 cells versus stable PC-3 transfectants expressing CA-ILK (PC-3/CA-ILK), and (B) SKBR3 cells versus transient SKBR3 transfectants expressing
ectopic CA/ILK (SKBR3/CA-ILK). Cells were treated with 22 at the indicated concentrations in 5% FBS-supplemented medium for 24 h. These data
are representative of three independent experiments. Blot on the left confirms the presence of the CA-ILK protein (*) in transfected SKBR3 cells.
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Figure 7. Evidence that 22 induces cell death through both apoptosis and autophagy. (A) Histograms depicting the percentages of PC-3 cells at various
phases of the cell cycle after exposure to the indicated concentrations 22 in 5% FBS-supplemented medium for 24 h. Each data point represents the mean
( SD (n = 3). (B) Dose-dependent effect of 22 on the extent of apoptosis induction, as detected by annexin V/PI staining. The values in the right upper
and lower quadrants denote the average of three independent experiments. (C) Dose-dependent effects of 22 on PARP cleavage and LC3-II conversion
in PC-3 cells after 24 h of treatment. (D) Protective effect of the stable transfection of CA-ILK (*), as depicted in Figure 4B, on the induction of LC3-II
conversion by 1 μM 22 in PC-3 cells. (E) siRNA-mediated knockdown of Atg5 inhibited 22-induced autophagy (left panel) as determined by LC3-II
conversion, and provided partial protection against 22-mediated suppression of PC-3 cell viability (right panel). Points, means; bars, SD (n = 6). All
immunoblots shown are representative of three independent experiments.
(FAK) at Tyr-397, a marker of FAK inhibition.30 In addition, as
evidence suggests the intermediary role of ILK in mediating
growth factor/integrin-induced activation of ERKs31ꢀ34 or
p3835ꢀ38 in various cell systems, we investigated the phosphor-
ylation status of ERKs and p38 versus JNKs in 22-treated cells.
As shown in Figure 6A, of the three MAP kinases evaluated,
22 showed dose-dependent suppressive effects on the levels of
phospho-ERK1/2 and phospho-p38, while that of phospho-JNK
remained unaltered. Equally important, stable expression of CA-
ILK prevented 22-facilitated inhibition of ERK and p38 phos-
phorylation, supporting the functional role of ILK in regulat-
ing the activation of ERKs and p38 in PC-3 cells (Figure 6B). In
contrast, CA-ILK showed no protective effect on the down-
regulation of S6 phosphorylation, confirming that 22 cross-
inhibited p70S6K.
Compound 22 Causes Cell Death through Autophagy and
Apoptosis. To shed light onto the mode of antiproliferative action
of 22, we assessed its effects on cell cycle progression and
programmed cell death (apoptosis versus autophagy) in PC-3 cells.
Flow cytometric analyses of cell cycle and Annexin V staining
indicate no apparent changes in cell cycle distribution or induction
of apoptosis until the 22 concentration exceeded a threshold of
2 μM (Figure 7A,B). Western blot analysis of PARP cleavage and
LC3-II conversion revealed that 22 induced both apoptosis and
autophagy and that the occurrence of drug-induced autophagy
preceded that of apoptosis in the doseꢀresponse relationship
(Figure 7C). As shown, 22-induced accumulation of LC3-II, an
essential step for autophagosome formation, was evident at
concentrations as low as 1 μM, while PARP cleavage occurred
at g2 μM. In addition, this induction of autophagy was blocked
by the stable expression of CA-ILK, suggesting that 22-induced
autophagy was attributable to ILK inhibition (Figure 7D).
Autophagy plays a dichotomous role in mediating cell fates,
either protective or destructive, in response to metabolic stress or
therapeutic agents.39 In this context, we examined the effect of
siRNA-mediated knockdown of autophagy-related 5 homologue
(Atg5) on 22-mediated suppression of PC-3 cell viability. As
shown, silencing of Atg5 disrupted 22-induced LC3-II proces-
sing and attenuated drug-induced cytotoxicity in PC-3 cells
(Figure 7E). This finding suggests that the induction of autop-
hagy represents a mechanism by which 22 mediates its anti-
proliferative activity, especially at low concentrations.
Suppressive Effect of Oral 22 on PC-3 Xenograft Tumor
Growth. The in vivo antitumor efficacy of 22 was evaluated in an
ectopic PC-3 tumor xenograft model. Athymic nude mice
bearing established subcutaneous PC-3 tumors were treated with
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role of ILK, reflected in the term of “ILK addiction”40 in many
types of cancers.16ꢀ22 Equally important, the ILK inhibitor 54
provides a proof-of-concept that ILK kinase activity can be
targeted to suppress tumor cell growth via inhibition of signaling
pathways mediated by Akt and YB-1.27,28,40 However, as recent
reports indicate that 54 as a single agent lacks in vivo antitumor
activity41 and that it is not specific for ILK,29 there is an urgency to
develop novel ILK inhibitors with higher potency and specificity.
Compound 22 was first identified through the screening of an
in-house focused compound library by immunoblotting against
Akt phosphorylation at Ser-473 versus Thr-308. Radiometric
assays using immunoprecipitated ILK from PC-3 cells demon-
strated the ability of 22 to inhibit ILK kinase activity with IC50 of
0.6 μM (Figure 4), which correlated with its high potency in
suppressing the phosphorylation levels of Ser-473-Akt and other
ILK substrates in cancer cells (Figure 3). Equally important,
22 exhibited a high degree of specificity against a panel of
recombinant kinases. Specifically, no appreciable inhibition by
22 was noted in a series of signaling kinases, including PDK1,
Akt, mTOR, GSK3β, FAK, cKit, EGFR, and FAK. An additional
line of evidence for the ILK-targeted activity of 22 was its
repressive effect on the mRNA and protein levels of the tran-
scription/translation factor YB-1 and its representative targets
HER2 and EGFR (Figure 5). As elevated expression and nuclear
localization of YB-1 is associated with increased proliferation,
multidrug resistance, tumor aggressiveness, and poor prognosis
in many types of cancers by stimulating the expression of a wide
array of growth-promoting genes,42 the ability of 22 to target YB-1
expression is noteworthy.
Evidence suggests that 22 mediates its antiproliferative effect
through the induction of both autophagy and apoptosis in PC-3
cells (Figure 7). Our data indicate that 22 induced autophagy
through ILK inhibition, which occurred at concentrations below
the threshold (2 μM) that our results show is required for
activating apoptosis in 22-treated cells. Moreover, as Atg5 silen-
cing attenuated autophagy and the suppressive effect of 22 on cell
viability (Figure 7E), this autophagy induction is integral to 22’s
antiproliferative activity, especially at low concentrations. Taken
together, this broad spectrum of mechanisms underlies the
therapeutic potential of 22 in cancer treatment, as manifested
by its in vivo efficacy as a single agent in suppressing PC-3
xenograft tumor growth (Figure 8).
Figure 8. In vivo antitumor efficacy of 22. (A) Effect of oral 22 on PC-3
xenograft tumor growth in athymic nude mice. Mice with established sc
PC-3 xenograft tumors were treated orally once daily with 22 at 25 and
50 mg/kg or vehicle for 35 days and tumor growth was monitored as
described in the Experimental Section. Points, mean; bars, SEM (n = 6).
(B) Western blot analysis of intratumoral biomarkers of drug activity in
three representative PC-3 tumors from each group of mice treated for 35
days as described above in (A).
oral 22 once daily at 25 and 50 mg/kg or the vehicle control. The
daily administration of 22 at both doses was well tolerated as the
mice showed no overt signs of toxicity or loss of body weight
(data not shown). Treatment with oral 22 in either dose resulted
in significant suppression (P < 0.05) of tumor growth relative
to the vehicle control after 35 days of treatment (48% and
62% suppression for 25 and 50 mg/kg, respectively; Figure 8A).
Examinations of intratumoral markers associated with drug
activity in three representative tumor lysates from each group
showed a dose-dependent inhibition of the phosphorylation of
Ser-473-Akt, while that of Thr-308-Akt was unaffected, accom-
panied by parallel decreases in the phosphorylation levels of
GSK3β and MLC and in the expression levels of YB-1, HER2,
and EGFR (Figure 8B). Together, the modulation of these
biomarkers validates ILK inhibition as at least part of the in vivo
mode of antitumor action of 22.
’ CONCLUSION
In conclusion, our data demonstrate that 22 is a novel, orally
bioavailable ILK inhibitor with a distinct mode of action that
inhibits tumor cell growth by modulating multiple signaling
pathways associated with oncogenesis and tumor progression.
Evaluation of the ability of 22 to block EMT and other aspects of
the aggressive phenotype and the use of 22 as a lead compound
for structural optimization to develop more potent ILK inhibi-
tors are underway.
’ DISCUSSION
Although substantial evidence has demonstrated the pivotal
role of ILK in regulating diverse cellular functions pertinent to
motility, proliferation, survival, and angiogenesis, a key issue that
remains in dispute is whether ILK possesses functional kinase
activity or serves as an adaptor protein to mediate these cellular
responses.12 Numerous studies, including the workdescribedhere,
have demonstrated that siRNA-mediated silencing or pharmaco-
logical inhibition of ILK in various types of normal and malignant
cells led to decreased phosphorylation of Ser-473-Akt and GSK3β.
Moreover, substantial evidence has demonstrated the oncogenic
’ EXPERIMENTAL SECTION
Chemical reagents and organic solvents were purchased from Sigma-
Aldrich (St. Louis, MO) unless otherwise mentioned. Nuclear magnetic
resonance spectra (1H NMR) were measured on a Bruker DPX 300
model spectrometer. Microwave reactions were carried out with the
CEM Discover SP-D pressurized microwave digestion system (CEM
Corp., Matthews, NC). Chemical shifts (δ) were reported in parts per
million (ppm) relative to the TMS peak. Electrospray ionization mass
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spectrometry analyses were performed with a Micromass Q-Tof II high-
resolution electrospray mass spectrometer in The Ohio State University
Campus Chemical Instrument Center. The purities of all tested com-
pounds are higher than 95% by elemental analyses, which were
performed by Atlantic Microlab, Inc. (Norcross, GA) and were reported
to be within 0.4% of calculated values. Flash column chromatography
was performed using silica gel (230ꢀ400 mesh). Compounds of series
AꢀC were synthesized according to slight modifications of previously
reported procedures.23,24 The general procedures for the synthesis of
compounds with carboxamide and propanamide side chains (series
DꢀF, 21 and 53) are described in Figure 2, which is illustrated by the
synthesis of the lead ILK inhibitor 22 as an example.
N-Methyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(40-(trifluoro-
methyl)-[1,10-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide
(22). Step a. (40-(Trifluoromethyl)- phenyl)boronic acid (4.7 g, 25 mmol)
was added to a solution of 4-bromo-acetophenone (5 g, 25.1 mmol, 1 equiv),
palladium(II) acetate (125 mg, 2 mol %), potassium carbonate (10.4 g,
75.3 mmol), and tetrabutylammonium bromide (10.5 g, 32.6 mmol). The
reaction mixture was added water (250 mL), heated to 60 °C with stirring
for 2 h under argon, cooled, diluted with water, and extracted with ethyl
acetate. The organic layer was dried and concentrated, and the residue
was purified by flash column chromatography (ethyl acetateꢀhexane,
3:7) to afford 1-(40-(trifluoromethyl)-[1,10-biphenyl]-4-yl)ethanone
(i) in quantitative yield. 1H NMR (300 MHz, CDCl3) δ 2.66 (s, 3H),
7.72 (m, 6H), 8.07 (d, J = 8.4 Hz, 2H).
Step c. To a solutionof compound I (6.6 g, 25 mmol) indrymethylene
chloride (250 mL) was added ethyl 4-(1H-benzo[d][1,2,3]triazol-1-yl)-
4-oxobutanoate (7.4 g, 30 mmol) and magnesium bromide ethyl etherate
(13 g, 50 mmol) under argon. The resulting solution was stirred under
argon for 10 min, added dropwise N,N-diisopropylethylamine (4.8 mL,
50 mmol), stirred for 16 h, and washed, in tandem, with 10% HCl
(150 mL ꢁ 1) and water (200 mL ꢁ 2). The organic phase was dried
and concentrated. The residue was purified by chromatography (ethyl
acetateꢀhexane, 9/1), followed by recrystallization in ethanol to give
(Z)-3-hydroxy-1-(40-(trifluoromethyl)-[1,10-biphenyl]-4-yl)oct-2-ene-
1,6-dione (ii) as white crystal (9.7 g, 99% yield). 1H NMR (300 MHz,
CDCl3) δ 1.31 (t, J = 7.2 Hz, 3H), 2.72 (t, J = 7.2 Hz, 2H), 2.85 (t, J = 7.2
Hz, 2H), 4.18 (q, J = 7.2 Hz, 2H), 6.26 (s, 1H), 7.68 (d, J = 8.1 Hz, 2H),
7.74 (m, 4H), 7.98 (d, J = 8.1 Hz, 2H), 15.82 (s, 1H).
CH2Cl2 to give 3-(1-(4-aminophenyl)-5-(40-(trifluoromethyl)-[1,10-
biphenyl]-4-yl)-1H-pyrazol-3-yl)-N-methylpropanamide (v) as white
crystal (460 mg, 99%). 1H NMR (300 MHz, CDCl3) δ 2.50 (t, J = 7.5
Hz, 2H), 2.69 (s, 3H), 2.91 (t, J = 7.5 Hz, 2H), 6.82 (d, J = 6.9 Hz, 2H),
7.19 (d, J = 8.4 Hz, 2H), 7.72 (m, 4H), 7.83 (m, 4H).
Step i. To a solution of compound v (140 mg, 0.3 mmol) in dry xylene
(10 mL) was added bis(2-chloroethyl)amine hydrochloride (62 mg,
0.36 mmol). The resulting mixture and heated to 170 °C with stirring for
20 h, cooled, and concentrated. The residue was purified by chroma-
tography (MeOHꢀCH2Cl2ꢀNH4OH, 2:98:0.1), followed by recrys-
tallization in ethyl acetate to give N-methyl-3-(1-(4-(piperazin-1-
yl)phenyl)-5-(40-(trifluoromethyl)-[1,10-biphenyl]-4-yl)-1H-pyrazol-3-
1
yl)propanamide (22) as white powder (80 mg, 50%). H NMR (300
MHz, CDCl3) δ 2.45 (dd, J = 8.0, 7.4 Hz, 2H), 2.78 (d, J = 4.8 Hz, 3H),
3.01 (d, J = 8.0 Hz, 2H), 3.06 (m, 4H), 3.21 (dd, J = 6.2, 3.6 Hz, 4H), 5.54
(bs, 1H), 6.54 (s, 1H), 6.98 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.8 Hz, 2H),
7.64 (d, J = 8.3 Hz, 2H), 7.71 (q, J = 8.6 Hz, 4H), 7.93 (d, J = 8.3 Hz, 2H).
HRMS exact mass of C30H30F3N5O (M + H+): 534.2480 amu; found,
534.2467 amu. Anal. Calcd: C 67.53, H 5.67, F 10.68, N 13.13. Found: C
67.17, H 5.61, F 10.36, N 12.95.
3-(5-(40-Cyano-[1,10-biphenyl]-4-yl)-1-(4-(piperazin-1-yl)-
phenyl)-1H-pyrazol-3-yl)-N-methylpropanamide (5). 1H NMR
(300 MHz, CDCl3): δ 2.47 (t, J = 7.5 Hz, 2H), 2.80 (d, J = 4.7 Hz, 3H),
3.02 (d, J = 8.2 Hz, 2H), 3.07 (dd, J = 3.9, 1.9 Hz, 4H), 3.27ꢀ3.18 (m,
4H), 5.45 (bs, 1H), 6.55 (s, 1H), 7.00 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.7
Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 7.73 (m, 4H), 7.95 (d, J = 8.4 Hz, 2H).
HRMS exact mass of C30H30N6O (M + H+): 491.2559 amu; found,
491.2546 amu.
N-Methyl-3-(5-(phenanthren-2-yl)-1-(4-(piperazin-1-yl)-
phenyl)-1H-pyrazol-3-yl)propanamide (14). 1H NMR (300
MHz, CDCl3): δ 2.49 (t, J = 7.5 Hz, 2H), 2.80 (d, J = 4.6 Hz, 3H),
3.05 (m, 6H), 3.21 (m, 4H), 5.49 (bs, 1H), 6.67 (s, 1H), 7.00 (d, J = 8.6
Hz, 2H), 7.39 (d, J = 8.6 Hz, 2H), 7.63 (m, 2H), 7.76 (q, J = 8.7 Hz, 2H),
7.89 (d, J = 7.7 Hz, 1H), 8.16 (dd, J = 8.4, 0.8 Hz, 1H), 8.35 (m, 1H), 8.71
(dd, J = 4.8, 3.7 Hz, 2H). HRMS exact mass of C31H31N5O (M + H+):
490.2607 amu; found, 490.2625 amu.
1-(4-(3-Aminopropanamido)phenyl)-N-methyl-5-(40-(tri-
fluoromethyl)-[1,10-biphenyl]-4-yl)-1H-pyrazole-3-carbox-
amide (15). 1H NMR (300 MHz, CDCl3) δ 2.84 (t, J = 5.6 Hz, 2H),
2.93 (s, 3H), 3.26 (d, J = 5.7 Hz, 2H), 7.02 (s, 1H), 7.33 (m, 4H), 7.68
(m, 6H), 7.80 (d, J = 8.0 Hz, 2H). HRMS exact mass of C27H24F3N5O2
(M + H+): 508.1960 amu; found, 508.1953 amu.
Step d. To a solution of compound ii (981 mg, 2.5 mmol) in ethanol
(10 mL) was added 4-nitro-phenylhydrazine hydrochloride (570 mg,
3 mmol) and 4-methylbenzene-sulfonic acid (430 mg, 2.5 mmol). The
resulting solution was stirred under microwave at 120 °C for 10 min,
concentrated, and recrystallized in ethanol to give ethyl ethyl 3-(1-(4-
nitrophenyl)-5-(40-(trifluoromethyl)-[1,10-biphenyl]-4-yl)-1H-pyrazol-
1-(4-(3-Aminopropanamido)phenyl)-N-ethyl-5-(40-(triflu-
oromethyl)-[1,10-biphenyl]-4-yl)-1H-pyrazole-3-carboxamide
(16). 1H NMR (300 MHz, CDCl3) δ 1.23 (t, J = 7.1 Hz, 3H), 2.82 (t, J =
6.0 Hz, 2H), 3.23 (m, 2H), 3.43 (dd, J = 14.1, 7.0 Hz, 2H), 7.04 (s, 1H),
7.41ꢀ7.29 (m, 4H), 7.76ꢀ7.62 (m, 6H), 7.81 (d, J = 8.2 Hz, 2H). HRMS
exact mass of C28H26F3N5O2 (M + H+): 522.2117 amu; found,
522.2112 amu.
1
3-yl)propanoate (iii) as yellow crystal (866 mg, 72% yield). H NMR
(300 MHz, CDCl3) δ 1.28 (t, J = 7.2 Hz, 3H), 2.77 (t, J = 7.5 Hz, 2H),
3.17 (t, J = 7.5 Hz, 2H), 4.18 (q, J = 7.2 Hz, 2H), 6.69 (s, 1H), 7.69 (d, J =
8.1 Hz, 2H), 7.74 (m, 4H), 7.81 (d, J = 8.4 Hz, 2H), 7.97 (d, J = 9.3 Hz,
2H), 8.41 (d, J = 8.4 Hz, 2H).
Step e. To a solution of compound iii (1 g, 2 mmol) in ethanol (2 mL)
was added 1 M methylamine in ethanol solution (5 mL). The resulting
solution was heated to 120 °C with stirring in a sealed tube for 16 h and
concentrated, followed by recrystallization in ethanol to give N-methyl-
3-(1-(4-nitrophenyl)-5-(40-(trifluoromethyl)-[1,10-biphenyl]-4-yl)-1H-
1-(4-(3-Aminopropanamido)phenyl)-N-isopropyl-5-(40-
(trifluoromethyl)-[1,10-biphenyl]-4-yl)-1H-pyrazole-3-car-
boxamide (17). 1H NMR (300 MHz, CDCl3) δ 1.18 (d, J = 6.3 Hz,
6H), 2.75 (t, J = 5.8 Hz, 2H), 3.06 (m, 2H), 4.20 (m, 1H), 7.04 (s, 1H),
7.41ꢀ7.29 (m, 4H), 7.76ꢀ7.62 (m, 6H), 7.81 (d, J = 8.2 Hz, 2H).
HRMS exact mass of C29H28F3N5O2 (M + H+): 536.2273 amu; found,
536.2269 amu.
3-Amino-N-(4-(3-(3-(methylamino)-3-oxopropyl)-5-(40-
(trifluoromethyl)-[1,10-biphenyl]-4-yl)-1H-pyrazol-1-yl)phe-
nyl)propanamide (18). 1H NMR (300 MHz, CDCl3) δ 2.51 (dd, J =
10.0, 5.6 Hz, 2H), 2.69 (s, 3H), 3.98 (m, 2H), 3.30 (m, 2H), 3.48 (m,
2H), 6.66 (s, 1H), 6.82 (d, J = 8.3 Hz, 2H), 7.19 (d, J = 8.5 Hz, 2H), 7.73
(m, 4H), 7.86 (m, 4H). HRMS exact mass of C29H28F3N5O2 (M + H+):
536.2273 amu; found, 536.2269 amu.
1
pyrazol-3-yl)propanamide (iv) as yellow powder (712 mg, 70%). H
NMR (300 MHz, CDCl3) δ 2.60 (t, J = 7.2 Hz, 2H), 2.80 (s, 3H), 3.21 (t,
J = 7.2 Hz, 2H), 5.45 (bs, 1H), 6.66 (s, 1H), 7.68 (m, 2H), 7.73 (m, 4H),
7.82 (d, J = 7.8 Hz, 2H), 7.96 (d, J = 6.9 Hz, 2H), 8.39 (d, J = 8.4 Hz, 2H).
Step f. To a solution of compound iv (500 mg, 1 mmol) in methanolꢀ
ethyl acetate (1:3, 10 mL) was added Pd on activated charcoal (15 mg).
The resulting mixture was stirred under H2(g) at 70 psi for 12 h, filtered,
and concentrated. The residue was then purified by chromatography
(MeOHꢀCH2Cl2ꢀNH4OH, 1:99:0.1), followed by recrystallization in
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Journal of Medicinal Chemistry
ARTICLE
3-Amino-N-(4-(3-(3-(ethylamino)-3-oxopropyl)-5-(40-(tri-
fluoromethyl)-[1,10-biphenyl]-4-yl)-1H-pyrazol-1-yl)phenyl)-
propanamide (19). 1H NMR(300MHz, CDCl3) δ 1.08 (t, J = 7.3Hz,
3H), 2.54 (t, J = 7.4 Hz, 2H), 2.88 (t, J = 6.3 Hz, 2H), 2.97 (t, J = 7.3 Hz,
2H), 3.18 (m, 2H), 3.30 (m, 2H), 6.74 (s, 1H), 7.50 (d, J = 8.5 Hz, 2H),
7.74 (d, J = 8.3 Hz, 4H), 7.84 (t, J = 7.1 Hz, 4H), 7.92 (d, J = 8.4 Hz, 2H).
HRMS exact mass of C30H30F3N5O2 (M + H+): 550.2430 amu; found,
550.2429 amu.
3-Amino-N-(4-(3-(3-(isopropylamino)-3-oxopropyl)-5-(400-
(trifluoromethyl)-[1,10-biphenyl]-4-yl)-1H-pyrazol-1-yl)phe-
nyl)propanamide (20). 1H NMR (300 MHz, CDCl3): δ 1.12 (d, J =
6.6 Hz, 6H), 2.56 (m, 2H), 2.91 (m, 2H), 2.99 (m, 2H), 4.95 (m, 1H),
6.77 (s, 1H), 7.53 (d, J = 8.7 Hz, 2H), 7.77 (d, J = 8.1 Hz, 4H), 7.87 (t, J =
7.4 Hz, 4H), 7.95 (d, J = 8.5 Hz, 2H). HRMS exact mass of
C31H32F3N5O2 (M + H+): 564.2586 amu; found, 564.2579 amu.
N-Ethyl-1-(4-(piperazin-1-yl)phenyl)-5-(40-(trifluoromethyl)-
[1,10-biphenyl]-4-yl)-1H-pyrazole-3-carboxamide (21). 1H NMR
(300 MHz, CDCl3) δ 1.26 (t, J = 7.1 Hz, 3H), 3.05 (m, 4H), 3.20 (m, 4H),
3.51 (m, 2H), 6.92 (d, J= 8.6 Hz, 1H), 7.00 (t, J= 5.4 Hz, 1H), 7.10 (s, 1H),
7.23 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 7.9 Hz, 2H), 7.55 (d, J = 7.8 Hz, 2H),
7.69 (s, 4H). HRMS exact mass of C29H28F3N5O (M + H+): 520.2324
amu; found, 520.2312 amu.
Lonza Biologics, Inc. (Hopkinton, MA) and maintained in the defined
growth medium obtained from the vendor. All cells were cultured at
37°C in a humidified incubator containing 5% CO2.
Transfection and Generation of Stable Sublines. Cancer
cells were transfected with plasmids or siRNA by electroporation using
the Cell Line Nucleofector Kit of the Amaxa Nucleofector system
(Lonza) according to manufacturer’s instructions. Transient transfec-
tants (expressing ILK shRNA, Atg5 siRNA, or GFP-CA-ILK [SKBR3])
were used for experiments 48 h after transfection. Stable PC-3 clones
expressing GFP-CA-ILK or GFP were selected after one week exposure
to G418 (250 μg/mL) by sorting for GFP signal on a FACSAria cell
sorter (BD Biosciences, Franklin Lakes, NJ).
Cell Viability Assay. Cell viability was determined using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay.
The assay was performed in 96-well plates in which cancer cells were
seeded at 5000 cells/well and nonmalignant cells at 8000 cells/well in
the presence of 10% FBS 24 h prior to treatment. Cells were then treated
with compounds for 24 h in the presence of 5% FBS. One-fourth volume
of medium containing a 5ꢁ concentration of MTT (0.25 mg/mL) was
added to each well followed by incubation at 37 °C for 1 h. After removal
of medium, the reduced MTT dye in each well was solubilized in 100 μL
of DMSO and absorbance was measured at 570 nm.
N-Ethyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(40-(trifluoro-
methyl)-[1,10-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide
(23). 1H NMR (300 MHz, CDCl3) δ 1.10 (t, J = 7.3 Hz, 3H), 2.44 (t, J =
7.7 Hz, 2H), 3.03 (m, 6H), 3.20 (m, 4H), 3.28 (m, 2H), 5.53 (bs, 1H),
6.54 (s, 1H), 6.98 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.8 Hz, 2H), 7.63 (d, J =
8.3 Hz, 2H), 7.71 (q, J = 8.6 Hz, 4H), 7.93 (d, J = 8.3 Hz, 2H). HRMS
exact mass of C31H32F3N5O (M + H+): 548.2637 amu; found, 548.2623
amu. Anal. Calcd: C 67.99, H 5.89, F 10.41, N 12.79. Found: C 67.67, H
5.83, F 10.16, N 12.63.
N-Methyl-3-(1-(4-morpholinophenyl)-5-(40-(trifluoro-
methyl)-[1,10-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide
(53). 1H NMR (300 MHz, CDCl3) δ 2.47 (t, J = 7.5 Hz, 2H), 2.78 (d, J =
4.8 Hz), 3.03 (m, 2H), 3.22 (m, 4H), 3.90 (m, 4H), 5.49 (bs, 1H), 6.54
(s, 1H), 6.98 (d, J = 8.7 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.1
Hz, 2H), 7.72 (q, J = 8.7 Hz, 4H), 7.94 (d, J = 8.1 Hz, 2H). HRMS exact
mass of C30H29F3N4O2 (M + Na+): 557.2140 amu; measured mass,
557.2144 amu.
Reagents and Antibodies. For in vitro studies, stock solutions of
the test agents were made in DMSO and diluted in culture medium to a
final DMSO concentration of 0.1%. For administration to nude mice,
agents were prepared as suspensions in sterile water containing 0.5%
methylcellulose and 0.1% Tween 80. The target proteins and commer-
cial sources of antibodies used in the study were as follows: Akt, p-473S-
Akt, p-308T-Akt, GSK-3β, p-9S-GSK-3β, MLC, p-18T/19S-MLC, LC3,
EGFR, ATG5, GFP, PCKR (Cell Signaling Technology, Inc., Beverly,
MA), p-422S-SGK1, YB-1 (Abcam, Inc., Cambridge, MA), p-657S-PCKR
(Millipore, Billerica, MA), HER2, c-Met, SGK1 (Santa Cruz Biotech-
nology, Inc., Santa Cruz, CA), β-actin (MP Biomedicals, Irvine, CA).
The shRNA for ILK in the pLKO.1 vector was purchased from Sigma-
Aldrich (St. Louis, MO). The human ILK full-length cDNA in the
pCMV-SPORT6 vector was purchased from Thermo Scientific (Rockford,
IL). GFP-tagged CA-ILK (constitutively active form S343D) was con-
structed from the human ILK full-length cDNA by site-directed mutagen-
esis and inserted into the pEGFP-C1 vector. Control siRNA and ATG5
siRNA were purchased from Cell Signaling Technology (Danvers, MA).
Cell Culture. All cancer cells were purchased from the American
Type Culture Collection (Manassas, VA). The PC-3 and LNCaP
prostate cancer cells were maintained in RPMI 1640 medium, MDA-
MB-231 and MDA-MB-468 breast cancer cells in DMEM medium, and
SKBR3 and MCF7 breast cancer cells in DMEM/F12 medium, all
supplemented with 10% FBS. Nonmalignant human epithelial cells of
the prostate (PrEC) and mammary gland (MEC) were obtained from
Immunoblotting. Treated cells were collected by scraping fol-
lowed by centrifugation, washed once with cold phosphate-buffered
saline (PBS), and then lysed in lysis buffer, consisting of 1% sodium
dodecyl sulfate (SDS), 10 mM ethylenediaminetetraacetic acid (EDTA),
and 50 mM Tris-HCl (pH 8.1) in the presence of a protease inhibitor
cocktail (Sigma-Aldrich). Lysates were sonicated for 10 s and then
centrifuged at 13200g for 15 min. Protein concentrations of the super-
natants were determined using a colorimetric bicinchoninic acid assay
(Pierce, Rockford, IL). An equivalent volume of 2ꢁ SDS-polyacryla-
mide gel electrophoresis sample loading buffer (300 mM Tris-HCl, pH
6.8; 10% SDS; 5% β-mercaptoethanol; 50% glycerol and 0.05% bromo-
phenol blue) was added to each sample, which were then was incubated
in boiling water for 10 min. Equal amounts of protein were resolved in
SDS-polyacrylamide gels in a minigel apparatus and then transferred to
nitrocellulose membranes. After blocking with Tris-buffered saline
containing 0.1% Tween 20 (TBST) and 5% nonfat milk for 40 min,
the membrane was washed three times with TBST for a total of 30 min
and then incubated with primary antibody at 1:1000 dilution in TBST at
4 °C for 2 h. The membrane was again washed three times with TBST for
a total of 30 min and then incubated with goat antirabbit or antimouse
immunoglobulin G-horseradish peroxidase conjugates (1:5000) for1 h at
room temperature. After a final three washes, the proteins were then
visualized by enhanced chemiluminescence.
RNA Extraction and RT-PCR. Total RNA was isolated from PC3,
PC-3/CA-ILK, SKBR3, and SKBR3/CA-ILK cells (1 ꢁ 106 cells) using
Trizol reagent (Invitrogen Corporation, Carlsbad, CA). Aliquots of 1 μg
of total RNA from each sample were reverse-transcribed to cDNA using
the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA).
PCR products were resolved by electrophoresis in 2% agarose gels and
visualized by ethidium bromide staining. The sequences of primers used
for RT-PCR were as follows.
YB-1: forward primer, 50-AAGTGATGGAGGGTGCTGAC-30; re-
verse primer 50-TTCTTCATTGCCGTCCTCTC-30. HER2: forward
primer, 50-TCCTGTGTGGACCTGGATGAC-30; reverse primer, 50-
CCAAAGACCACCCCCAAGA-30. EGFR: forward primer, 50-GGAC-
GACGTGGTGGATGCCG-30; EGFR: reverse primer, 50- GGCGCC-
TGTGGGGTCTGAGC-30. β-Actin: forward primer, 50- CACGAAAC-
TACCTTCAACTCCA-30; reverse primer, 50- GAAGCATTTGCG-
GTGGACGAT-30.
Radiometric ILK Kinase Assay. The kinase activity of immuno-
precipitated ILK was determined in an in vitro radiometric kinase assay
using myelin basic protein (MBP) as substrate and [γ-32P] ATP as
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Journal of Medicinal Chemistry
ARTICLE
phosphate donor according reported procedures6,8 with modifica-
tions. For immunoprecipitation, PC-3 cells were pretreated with EGF
(100 ng/mL) for 2 h and then lysed (5 ꢁ 105 /sample) in 250 μL of lysis
buffer (20 mM Tris-HCl [pH 7.5), 150 mM NaCl, 1% Triton X-100)
for 2 h on ice. After sonication of lysates and centrifugation (13200g,
15 min), aliquots of the supernatants were combined with 3 μL ILK
antibody and 15 μL of protein A/G PLUS-agarose (Santa Cruz). Imm-
unocomplexes containing immunoprecipitated ILK (A/G PLUS-ILK)
were then collected by centrifugation. Kinase activity assays were
performed in 25 μL kinase reaction buffer (Cell Signaling) containing
5 μg of MBP and 2.5 μCi [γ-32P] ATP (3000 Ci/mmol, 10mCi/ml;
Perkin-Elmer, Waltham, MA), with and without 15 μL of A/G PLUSꢀ
ILK. Reactions were incubated at 30 °C for 25 min and stopped by
addition of SDS-PAGE sample buffer. Proteins were resolved on 15%
SDS-polyacrylamide gels and [γ-32P] MBP was detected by autoradio-
graphy. Densitometric analysis was performed to determine the relative
intensities in drug-treated samples versus those in the respective DMSO-
treated controls.
Flow Cytometry. After 48 h treatment with 22, both adhered and
unattached PC-3 cells were harvested, resuspended in ice-cold PBS (1 ꢁ
106 cells/200 μL), and then fixed with 1 mL of 100% ethanol. For cell
cycle analysis, fixed cells were pelleted, resuspended in 500 μL of
propidium iodide (PI) staining solution (80 μg/mL PI, 100 μg/mL
RNase, 0.1%, v/v, Triton X-100 in PBS) at 37 °C for 30 min in darkness,
and then filtered through a 40 μm nylon mesh. For assessment of
apoptosis, cells were costained with FITC-conjugated annexin V and PI
according to manufacturer’s instructions. The distribution of cells among
the phases of the cell cycle and numbers of apoptotic cells were
determined using a FACSCalibur (BD Biosciences) flow cytometer
and FlowJo software (Tree Star, Inc., Ashland, OR) for analysis.
PC-3 Xenograft Tumor Model. Xenograft tumors were estab-
lished in male NCr athymic nude mice (5ꢀ7 weeks of age; National
Cancer Institute, Frederick, MD) by subcutaneous injection of 0.5 ꢁ 106
PC-3 cells in a total volume of 0.1 mL of serum-free medium containing
50% Matrigel (BD Biosciences). Mice with established tumors (157.1 (
29.1 mm3) were randomized to three groups (n = 6) receiving single
daily treatments of 22 at 25 or 50 mg/kg or vehicle (0.5% methylcellu-
lose, 0.1% Tween 80 in water) for 35 days by oral gavage at a volume of
10 μL/g body weight. Tumor volumes were calculated from weekly
caliper measurements using a standard formula (volume = width2 ꢁ
length ꢁ 0.52). Body weights were measured weekly. At terminal
sacrifice, tumors were harvested, snap-frozen in liquid nitrogen, and
stored at ꢀ80 °C until used for analysis of biomarkers associated with
ILK inhibition. All experimental procedures using live animals were
conducted in accordance with protocols approved by The Ohio State
University Institutional Animal Care and Use Committee.
Research. We thank Trent Godard for technical assistance in the
synthesis of 22.
’ ABBREVIATIONS USED
ILK, integrin-linked kinase; PI3K, phosphatidylinositol-3-kinase;
PIP3, phosphatidylinositol 3,4,5-trisphosphate; PDK, phosphoi-
nositide-dependent kinase; mTOR, mammalian target of rapa-
mycin; mTORC2, the rictor-mTOR complex; GSK3β, glycogen
synthase kinase 3β; MLC, myosin light chain; SGK-1, serum- and
glucocorticoid-induced protein kinase 1; PKCR, protein kinase
C R;MBP myelin basic protein; CA, constitutively active; FAK,
focal adhesion kinase
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Statistical Analysis. Quantitative data from in vitro experiments
arepresented as mean ( SD. Data frominvivo experiments areexpressed
as mean ( SEM. Differences among group means were analyzed for
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: (614)-688-4008. Fax: (614)-688-8556. E-mail: chen.844@
osu.edu.
’ ACKNOWLEDGMENT
(14) Deng, J. T.; Sutherland, C.; Brautigan, D. L.; Eto, M.; Walsh,
M. P. Phosphorylation of the myosin phosphatase inhibitors, CPI-17
and PHI-1, by integrin-linked kinase. Biochem. J. 2002, 367, 517–524.
This work is supported by National Institutes of Health grant
CA112250 and the Stephanie Spielman Fund for Breast Cancer
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